This invention was made with U.S. Government support under Contract No. GM 37277 awarded by the National Institute of General Medical Sciences of the United States Department of Health and Human Services.
This invention relates a method for characterizing polymer molecules or the like, for example, observing and determining the size of individual particles and determining the weight distribution of a sample containing particles of varying size. More particularly, this invention involves the use of microscopy and/or microscopy in combination with spectroscopic methods to characterize particles, such as by measuring their positional and conformational changes when they are subjected to an external force, and by measuring their length and diameter or radius. Such measurements include rates of relaxation, reorientation and rotation of particles subject to an external force, and measurements of length and diameter of particles before, during or after they are subjected to an external force.
Among other applications, these measurements are used to determine particle size. The invention is especially well suited to size polymer molecules in a polydisperse sample (e.g., a sample containing particles of varying size), when the particles have been placed in some type of medium, and is useful to measure very large molecules, such as large nucleic acid molecules which are subject to breakage when placed on a microscope slide using conventional methods.
Methods for determining the molecular weight distribution of a polydisperse sample of particles are useful in a variety of different fields. For example, in polymer chemistry, the properties of an oligomer are often dependent upon its molecular weight distribution. When a particular substance is found to exhibit favorable properties and the exact composition of the oligomer is not known, an analysis of the molecular weight distribution of the polymer is used for purposes of identification. In molecular biology, the molecular weight distribution of a polydisperse sample, such as a sample of DNA restriction enzyme digests, provides valuable information about the organization of the DNA. This information may be used to produce chromosome maps and extensive molecular genetics characterizations.
Traditionally, the molecular weight distribution of a sample of particles has been determined by measuring the rate at which particles which are subjected to a perturbing force move through an appropriate medium, e.g., a medium which causes the particles to separate according to size. A mathematical relationship is calculated which relates the size of particles and their migration rate through a medium when a specified force is applied. For example, in gel permeation chromatography, a well-known technique, a polymer to be characterized is dissolved in a solvent and the resulting solution is then passed through a column which has a cross-linked or porous gel polymer in the stationary phase. Large molecules will pass quickly through the gel, while the movement of smaller molecules will be hindered by their entry into the pores of the substance comprising the stationary phase. The molecular weight distribution of the sample is determined by measuring the content of the effluent from the column, e.g., by measuring the refractive index of the effluent over a period of time. Several limitations of gel permeation chromatography are that it cannot be used to separate DNA molecules larger than about 5 kb, and it can only be used for samples which are soluble in (at least one of) a limited number of suitable solvents.
Sedimentation is a well-known technique for measuring particle size, but, when applied to polymers, this method is limited to molecules with a maximum size of about 50-100 kilobases (kb). Attempting to measure larger molecules by this technique would probably result in underestimation of molecular size, mainly because the sedimentation coefficient is sensitive to centrifuge speed. (see Kavenoff et al., Cold Spring Harbor Symp. Quantit. Biol., 38, 1 (1974).
Another popular method of separating polymer particles by size is by gel electrophoresis (see, e.g., Freifelder, Physical Biochemistry, W. H. Freeman (1976), which is particularly useful for separating restriction digests. In brief, application of an electric field to an agarose or polyacrylamide gel in which polymer particles are dissolved causes the smaller particles to migrate through the gel at a faster rate than the larger particles. The molecular weight of the polymer in each band is calibrated by a comparison of the migration rate of an unknown substance with the mobility of polymer fragments of known length. The amount of polymer in each band can be estimated based upon the width and/or color intensity (optical density) of the stained band, however, this type of estimate is usually not very accurate.
Pulsed field electrophoresis, developed by the present inventor and described in U.S. Pat. No. 4,473,452, the disclosure of which is hereby incorporated by reference and relied upon, is an electrophoretic technique in which the separation of large DNA molecules in a gel is improved relative to separation using conventional electrophoresis. According to this technique, deliberately alternated electric fields are used to separate particles, rather than the continuous fields used in previously known electrophoretic methods. More particularly, particles are separated using electric fields of equal strength which are transverse to each other, which alternate between high and low intensities out of phase with each other at a frequency related to the mass of the particles. The forces move the particles in an overall direction transverse to the respective directions of the fields. It should be noted here that the term "transverse" as used herein is not limited to an angle of, or close to, 90.degree., but includes other substantial angles of intersection.
One of the most significant problems with determining the weight of molecules by indirect measurement techniques, such as those described above, is that the parameters which are directly measured, e.g., migration rate, are relatively insensitive to small differences in molecular size. Thus, a precise determination of particle size distribution is difficult to obtain. The lack of precision may particularly be a problem when biological polymer samples, which tend to be unstable and contain single molecules inches in length, are involved.
While some of the known methods of determining particle size distribution in a polydisperse sample provide better resolution than others, few, if any, of the previously known techniques provide resolution as high as is needed to distinguish between particles of nearly identical size. Gel permeation chromatography and sedimentation provide resolution of only about M.sup.1/2 (M=molecular weight). Standard agarose gel electrophoresis and polyacrylamide gel electrophoresis provide resolution varying as -logM. Pulsed electrophoretic techniques are effective for separating extraordinarily large molecules, but do not provide much better resolution than standard electrophoresis. Thus, the ability to distinguish between particles of similar size, for example, particles differing in length by a fraction of percent, is quite limited when the above-described measurement techniques are used.
Under special experimental circumstances, DNA gel electrophoresis resolves a polymer mixture to a resolution of M.sup.1. However, this degree of accuracy is only achieved when variables such as gel concentration and field strength are carefully controlled.
Particles of higher mass (i.e., up to approximately 600 kb) can be resolved using conventional gel electrophoresis by reducing the gel concentration to as low as 0.035% and reducing field strength, however, there are drawbacks to this method. Most notably, the dramatic reduction in gel concentration results in a gel which is mechanically unstable, and less sample can be loaded. An electrophoretic run to resolve very large DNA molecules using a reduced gel concentration and field strength may take a week or more to complete. Furthermore, a reduced gel concentration is not useful to separate molecules in a sample having a wide range of particle sizes, because separation of small molecules is not achieved. Thus, if a sample containing molecules having a wide range of sizes is to be separated, several electrophoretic runs may be needed, e.g., first, a separation of the larger molecules and then further separation of the smaller molecules.
Other particle measurement techniques known in the art are useful for sizing certain molecules which are present in a bulk sample, (e.g., the largest molecules in the sample, or the average molecular size) but are impractical for measuring many polymers of varying length in a given sample. The viscoelastic recoil technique, (see Kavenoff et al, "Chromosome-sized DNA molecules from Drosophila," Chromosoma 41, 1 (1973)) which is well known in the art, involves stretching out coiled molecules in a solvent flow field (e.g., a field which is created when fluid is perturbed between two moving plates) and determining the time required for the largest molecule to return to a relaxed state. Relaxation time is measured by watching the rotation of a concentric rotor which moves during the time of relaxation. While this technique is quite precise in that sample determinations vary as M.sup.1.66 when applied to large DNA molecules, it is not useful for sizing molecules other than the largest molecule in the sample.
Using light scattering techniques, which are known in the art, (e.g., quasi-elastic light scattering), the size and shape of particles are determined by a Zimm plot, a data analysis method which is known in the art. With these techniques, size dependence varies as M.sup.1. Light scattering requires that the solution in which the molecules to be measured are placed is pure, that is, without dust or other contamination, and it is therefore unsuitable for sizing a DNA sample. Furthermore, it is not useful for sizing molecules as large as many DNA molecules, and is useful only for determining the average weight of particles in a sample, not the weight distribution of a sample with particles of various sizes.
Yet another particle measuring technique which is known in the art for measuring individual molecules provides measurements of particle size having limited accuracy. The average size and shape of individual, relaxed DNA molecules has been determined by observing the molecules under a fluorescence microscope, and measuring the major and minor axes of molecules having a spherical or ellipsoid shape (see Yanagida et al, Cold Spring Harbor Symp. Quantit. Biol. 47, 177, (1983)). The technique described in the above-cited reference is performed in a free solution, without perturbation of the molecules.
The movement of small DNA molecules during electrophoresis has been observed (see Smith et al. Science, 243, 203 (1989)). The methods disclosed in this publication are not suitable for observation of very large DNA molecules, and techniques for measuring molecules are not discussed.
It is noted that practical weight determinations of particles such as polymer molecules depend not only upon maximizing the size dependencies of the directly measured parameters, but also upon factors such as the amount of sample needed, the time required to complete an analysis, and the accuracy of measurements. Gel permeation chromatography can be time-consuming and requires a large amount of sample. Methods such as conventional gel electrophoresis can be relatively time-consuming, require moderate amounts of sample, and cannot size very large DNA molecules.